WK-13-3D inhibits triple-negative breast cancer by blocking autophagic flux via AKT/mTOR inhibitio

Introduction and study purpose

This study assessed the antitumor potential and mechanism of action of the antimicrobial peptide WK-13-3D in models of triple-negative breast cancer (TNBC). The authors aimed to determine whether WK-13-3D impairs TNBC cell growth and clonogenicity and to elucidate molecular mechanisms with emphasis on autophagy-lysosome homeostasis, the AKT/mTOR signaling axis, and binding immunoglobulin protein (BiP). The work included in vitro experiments in established TNBC cell lines and in vivo validation in a nude mouse xenograft model.

Methods and experimental models

TNBC cell lines MDA-MB-231 and MDA-MB-468 were used as in vitro systems. Cell viability and clonogenic potential were assessed with CCK-8 and colony formation assays, respectively. Autophagy-related protein levels (LC3-II/I ratio, p62) and phosphorylation status of components in the AKT/mTOR pathway were measured by Western blot. Autophagosome formation was examined by transmission electron microscopy and immunofluorescence. Autophagic flux was monitored using an mCherry-GFP-LC3 tandem fluorescent adenoviral reporter. Lysosomal acidification was evaluated using LysoTracker Red and acridine orange (AO) staining. Functional rescue experiments employed the AKT activator SC79 and overexpression of BiP. An in vivo nude mouse xenograft model was used to validate antitumor activity of WK-13-3D.

In vitro antitumor effects of WK-13-3D

Treatment with WK-13-3D markedly suppressed viability of MDA-MB-231 and MDA-MB-468 cells and reduced their clonogenic potential, as measured by CCK-8 and colony formation assays. The peptide produced phenotypic effects consistent with reduced proliferative capacity in these TNBC cell models.

WK-13-3D effects on AKT/mTOR signaling and ER stress via BiP binding

Mechanistically, WK-13-3D concurrently inhibited the AKT/mTOR signaling pathway and directly bound to BiP, a principal endoplasmic reticulum (ER) chaperone. Binding to BiP was associated with induction of ER stress. The coordinated inhibition of AKT/mTOR signaling and engagement of BiP represent dual molecular actions through which WK-13-3D exerts downstream effects on autophagy-lysosome homeostasis.

Impact on autophagy: autophagosome accumulation and p62 degradation

WK-13-3D treatment led to a blockade of autophagic flux, evidenced by accumulation of autophagosomes and an increased LC3-II/I ratio. Additionally, degradation of the autophagic substrate p62 was impaired, consistent with disrupted completion of the autophagy process. Autophagosome formation was visualized by transmission electron microscopy and immunofluorescence, and flux was interrogated using the mCherry-GFP-LC3 tandem reporter.

Lysosomal function and acidification findings

Despite the observed autophagic flux blockade, lysosomal acidification and hydrolytic function were reported as not compromised following WK-13-3D treatment. This conclusion was based on LysoTracker Red and acridine orange staining, which indicated preserved lysosomal acidification. Thus, the autophagy blockade appeared to occur without overt loss of lysosomal acidity or gross hydrolytic dysfunction.

Functional rescue experiments: AKT activation and BiP overexpression

To dissect contributions of the identified pathways, the AKT activator SC79 was used in rescue experiments. Activation of AKT partially reversed the autophagy dysregulation induced by WK-13-3D, implicating AKT/mTOR inhibition as a mechanistic component of the peptide’s effect on autophagic flux. Separately, overexpression of BiP restored autophagic flux and enhanced lysosomal activity in the context of WK-13-3D exposure, supporting BiP as a direct molecular target whose modulation influences ER stress and lysosome-related processes. These functional perturbations indicate that both AKT/mTOR signaling and BiP-mediated ER homeostasis contribute to the peptide’s antitumor mechanism.

In vivo antitumor validation in a nude mouse xenograft model

The antitumor activity of WK-13-3D observed in vitro was validated in a nude mouse xenograft model. The manuscript reports that WK-13-3D exhibited in vivo antitumor effects in this model, supporting translational relevance of the observed cellular mechanisms. Specific in vivo dosing, tumor growth curves, or statistical details were not provided in the abstract and therefore are not reported here.

Conclusions, implications, and next steps

The authors conclude that WK-13-3D disrupts autophagy-lysosome crosstalk in TNBC by coordinated inhibition of AKT/mTOR signaling and direct targeting of BiP, leading to autophagic flux blockade and induction of ER stress. These dual-targeting effects produced a potent antitumor response in TNBC models and position WK-13-3D as a promising lead candidate for further preclinical investigation. The abstract indicates that these mechanistic insights may inform future development of therapeutics that exploit autophagy-lysosome and ER stress pathways in TNBC.

Note: The abstract reports the main experimental approaches, key mechanistic findings, and in vivo validation. Detailed experimental parameters, quantitative results, and full methodological particulars are contained in the full text and were not reported within the abstract.